Abstract: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. Most cases of HCC develop in patients that already have liver cirrhosis and have been recommended for surveillance for an early onset of HCC. Only 20-30% of patients with HCC are currently eligible for curative therapy with surgical resection where the impact of these therapeutic interventions on the overall survival of patients with HCC is related to the detection of HCC at earlier stages. With the development of new markers one would hope to increase the proportion of patients that can be offered curative therapies and significantly impact the survival of patients with this disease. In our previous work we have found several glycoprotein markers of early stage HCC versus cirrhosis which can provide a performance that is potentially better than that of AFP or together with AFP can improve its overall performance. In particular a bifucosylated glycan structure found in haptoglobin from HCC patients is undergoing a phase 2 EDRN blinded validation where this marker appears to improve the performance for detection of early stage HCC of all etiologies. A new strategy that has been investigated is to screen serum for large numbers of core-fucosylated(CF) sites using mass spectrometry where over 1300 sites have been detected in the serum of HCC patients. We have found several CF-glycopeptides in serum that can distinguish between early HCC and cirrhosis and which are etiology specific for HCV-HCC versus HCV-cirrhosis and ALC-HCC versus ALC-cirrhosis. In the work proposed herein, we will develop a mass spec based assay that is specific for detecting candidate CF- glycopeptides that are differentially expressed in serum of early HCC patients versus cirrhosis. It involves digestion of proteins in total serum to glycopeptides followed by enzymatic removal of the glycan down to the GlcNAc-Fuc core fucosylation. The CF-glycopeptides are labeled with isotopic tags and analyzed by a novel glyco-MRM(multiple reaction monitoring) assay using tandem mass spectrometry where differences in CF expression between HCC and cirrhosis can be detected. This method can be multiplexed for many markers simultaneously, does not require the use of antibodies and can be automated. The hypothesis here is that these structural changes depend on a different mechanism than protein level assays and could provide complementary information to the ELISA assay for AFP where together they may provide an improved AUC. We will test these markers in a confirmation set of 300 samples and evaluate their performance together with the ELISA assay for AFP and a mass spec assay for bifucosylated Hp. The markers that are found to improve on the performance of AFP either alone or in combination with AFP or the bifucosylated Hp assay will then undergo a blinded validation in the phase 2 EDRN validation set. Markers that perform successfully in this blinded validation will then go on for possible use in future work in the HEDS study which is a large scale prospective-retrospective evaluation of markers for early detection of HCC.